Disease-Associated Mosaic Variation in Clinical Exome Sequencing: a Two Year Pediatric Tertiary Care

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TITLE PAGE

Disease-Associated Mosaic Variation in Clinical Exome Sequencing: A Two Year Pediatric Tertiary Care

Experience

Cecelia R. Miller1,2, Kristy Lee1,2, Ruthann B. Pfau1,2,3, Shalini C. Reshmi1,2,3, Donald J. Corsmeier1,

Sayaka Hashimoto1, Ashita Dave-Wala1, Vijayakumar Jayaraman1, Daniel Koboldt1,3, Theodora

Matthews1, Danielle Mouhlas1, Maggie Stein1, Aimee McKinney1, Tom Grossman1, Benjamin J. Kelly1,

Peter White1,3, Vincent Magrini1,3, Richard K. Wilson1,3, Elaine R. Mardis1,3, Catherine E. Cottrell1,2,3.

1 The Steve and Cindy Rasmussen Institute for Genomic Medicine, Nationwide Children’s Hospital,

Columbus, Ohio, USA; 2Department of Pathology, The Ohio State University, Columbus, Ohio, USA;

3Department of Pediatrics, The Ohio State University, Columbus, Ohio, USA;

Corresponding author:

Catherine E. Cottrell

[email protected]

Running title: Mosaicism in clinical exome sequencing

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ABSTRACT

Exome sequencing (ES) has become an important tool in pediatric genomic medicine, improving identification of disease-associated variation due to assay breadth. Depth is also afforded by ES, enabling detection of lower frequency mosaic variation compared to Sanger sequencing in the studied tissue, thus enhancing diagnostic yield. Within a pediatric tertiary care hospital, we report two years of clinical ES data from probands evaluated for genetic disease to assess diagnostic yield, characteristics of causal variants, and prevalence of mosaicism amongst disease-causing variants. Exome derived, phenotype-driven variant data from 357 probands was analyzed concurrent with parental ES data, where available. Blood was the source of nucleic acid. Sequence read alignments were manually reviewed for all assessed variants. Sanger sequencing was employed for suspected de novo or mosaic variation.

Clinical provider notes were reviewed to determine concordance between laboratory-reported data and the ordering provider’s interpretation of variant-associated disease causality. Laboratory-derived diagnostic yield and provider-substantiated diagnoses had 91.4% concordance. The cohort returned 117 provider-substantiated diagnoses among 115 probands for a diagnostic yield of 32.2%. De novo variants represented 64.9% of disease-associated variation within trio analyses. Among the 115 probands, 5 harbored disease-associated somatic mosaic variation. Two additional probands were observed to inherit a disease-associated variant from an unaffected mosaic parent. Among inheritance patterns, de novo variation was the most frequent disease etiology. Somatic mosaicism is increasingly recognized as a significant contributor to genetic disease, particularly with increased sequence depth attainable from ES.

This report highlights the potential and importance of detecting mosaicism in ES.

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INTRODUCTION

Massively parallel sequencing (MPS) of the exome has demonstrated significant diagnostic and clinical utility in patients with suspected genetic disorders (Clark et al. 2018; Lee et al. 2014; Farwell et al. 2015;

Meng et al. 2017; Hu et al. 2018). Molecular diagnostic rates from clinical testing by hospital and reference laboratories performing exome sequencing (ES) range from 24-58%, likely influenced by laboratory-specific methodologies and by the characteristics of the patient population being evaluated

(Clark et al. 2018). Given the utility of ES, it has been proposed that the assay should be considered as a first-tier molecular test (Stark et al. 2016; Hu et al. 2018). An additional advantage to this type of testing is the ability to re-evaluate existing sequence data as advances in bioinformatic processing and variant detection occur over time. Furthermore, this approach allows for a consideration of the evolution of patient phenotype, in addition to the inclusion of updated/emerging data for disease-gene associations,

thus further enhancing diagnostic potential (Nambot et al. 2018; Ewans et al. 2018).

The increased utilization of MPS, including ES, has expanded our understanding of the

contribution of somatic mosaicism in genetic disorders by enhancing our ability to identify disease-

associated variants at low variant allele frequency/fraction (VAF). Genetic variation acquired during

embryogenesis and resulting in the establishment of two or more genetically distinct cell populations

represents post-zygotic mosaicism. Disease-causing variants can be confined to the germline (gonadal

mosaicism), resulting in disease when passed on to subsequent offspring. Alternatively, a genetic variant

can occur in the soma of a developing embryo (somatic mosaicism), with variable levels of the variant throughout the body dependent on cell lineage. In addition, a mosaic variant affecting both the soma and germline is referred to as gonosomal mosaicism (Biesecker and Spinner 2018). The phenotypic spectrum of post-zygotic somatic mosaicism can vary and is dependent upon timing of the manifestation of the variant during development, along with the proportion of cells harboring the variant and distribution across tissue types. Pathogenic variants at very low VAF in affected tissue can be sufficient to cause disease.

For example, in diseases such as Sturge-Weber, or vascular anomalies with overgrowth (e.g. Proteus syndrome or PIK3CA Related Overgrowth Spectrum (PROS)), the VAF of pathogenic variants in affected

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tissue has been reported as low as 1% (Hucthagowder et al. 2016; Shirley et al. 2013; Lindhurst et al.

2011).

Among unselected clinical exome cohort studies of pediatric, and combined pediatric and adult populations, disease-associated mosaic variants were noted at a frequency of approximately 1-1.5%

(Cao et al. 2019; Retterer et al. 2016; Yang et al. 2013). The frequency of mosaicism increases when examining for specific phenotypes. For example, in epilepsy-related neurodevelopmental disorders, 3% of the pathogenic variants identified by either an MPS epilepsy panel or ES were mosaic (Stosser et al.

2018). In certain disorders, for example McCune-Albright and PROS, mosaic variants are the primary

mechanism of disease (Hucthagowder et al. 2016; Aldred and Trembath 2000; Keppler-Noreuil et al.

2015).

We evaluated two years of clinical ES data from our laboratory within a pediatric tertiary care center to determine the characteristics of disease-associated variants within our cohort, as well as to compare the diagnostic yield reported by the laboratory versus the ordering clinical provider’s

interpretation of laboratory reported variant causality. We sought to evaluate the concordance of the

molecular ES diagnostic rate generated by the laboratory with clinical provider-confirmed diagnoses

recorded in the electronic medical record (EMR) to test if the laboratory workflow, including selection of

genes relevant to the proband phenotype and subsequent variant assessment, resulted in meaningful

results being reported back to the ordering provider. We further summarized the characteristics of these

provider-confirmed causal variants and evaluated the contribution of mosaic variants to genetic disease within the context of these diagnoses.

RESULTS

Patient cohort

In a consecutive 24-month period, proband (n=357) and parental (when available, n=601) peripheral blood samples were submitted for ES to The Steve and Cindy Rasmussen Institute for Genomic Medicine at Nationwide Children’s Hospital, Columbus, Ohio. Two submitted parental samples were excluded due to non-paternity. Cases represented 267 (74.8%) trio analyses of the proband plus both parental

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samples, 65 (18.2%) duo analyses of the proband and one parental sample, and 25 (7%) proband-only

analyses. All cases were reviewed or referred by a clinical geneticist at the time of test order. The cohort

consisted primarily of pediatric probands, or young adults with symptoms that initially presented in

childhood (average age = 7.2 years, range 0-56 years). A phenotype-informed approach was applied for tertiary analysis. The top 20 human phenotype-ontology (HPO) terms representing the most common clinician-provided phenotypic characteristics among probands in this population are shown in Table 1.

Consistent with other ES cohorts, global developmental delay (73.4% of our cohort), abnormal facial shape (51.3%), and muscular hypotonia (51.0%) were the most frequently described features (Farwell et al. 2015; Lee et al. 2014; Yang et al. 2014).

Genomic analyses

The clinical laboratory reported variants as likely causal for the proband’s phenotype for 128 genetic disorders among 123 probands (34.5%; 95% CI, 29.5%-39.6%). Ordering provider documentation in the

EMR corroborated 115 instances of the variant(s) being attributed to the etiology of the proband phenotype for a provider-substantiated diagnostic yield of 32.2% (95% CI, 27.4%-37.3%). Two probands were found to have two separate genetic disorders, for a total of 117 clinically confirmed genetic diagnoses. Clinical laboratory diagnoses and provider-substantiated diagnoses had an overall concordance of 91.4% [95% CI, 85.1%-95.6%]. Of the 11 laboratory reported genetic disorders not substantiated by clinician data in the EMR, 5 were due to insufficient overlap of features, 4 remained in the differential for further clinical evaluation as the proband features develop, and 2 had discordant inheritance patterns among affected/unaffected family members. A diagnosis was determined for 10/25

(40.0%) of the proband-only analyses, 10/65 (15.4%) duo analyses, and 95/267 (35.6%) trio analyses.

The patterns of inheritance for the 117 diagnoses included 75 (64.1%) autosomal dominant, 25 (21.4%) autosomal recessive and 17 (14.5%) X-linked (Fig. 1).

Out of the 916 individuals consented for medically actionable findings, 29 individuals (3.2%) had variants meeting ACMG/AMP criteria supportive of a Pathogenic/Likely Pathogenic classification. 8 families demonstrated a proband-parent shared finding, while 4 probands and 9 parents harbored events individually. Diseases represented by these findings were Loeys-Dietz syndrome (4%), malignant

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hypothermia susceptibility (10%), hypertrophic cardiomyopathy (10%), familial hypercholesterolemia

(14%), arrhythmogenic right ventricular cardiomyopathy (14%), long QT syndromes (24%), and hereditary

breast and ovarian cancer (24%).

Among 95 diagnostic ES trio studies, we identified 97 genetic disorders, of which the inheritance

patterns were autosomal dominant (n=62; 63.9%), autosomal recessive (n=21; 21.7%), and X-linked

(n=14; 14.4%). Among the autosomal dominantly-inherited diseases, 54 variants (87.1%) were confirmed as de novo; while 9 (64.3%) of the X-linked inherited disorders were attributable to de novo variation.

Overall, de novo variation accounted for 64.9% [63/97, 95% CI, 54.6%-74.4%] of the inheritance pattern of genetic disorders detected in trio ES.

Of the 117 disorders, genetic disease associations were diverse among this cohort with 65% of the identified disease-associated genetic loci unique to a single proband. Recurrently involved genetic loci included CREBBP, in which causal variants associated with Rubenstein-Taybi syndrome were identified in 3 probands. Additionally, 4 probands each had causal variants in ANKRD11 and IQSEC2, associated with KBG syndrome and X-linked intellectual disability, respectively. Causal variants of provider- substantiated diagnoses are provided in Table 2 and Table 3, with the most common HPO terms describing this cohort detailed in Table 1.

The proportion of the causal variant allele was documented for proband and parental samples from all cases with a provider-substantiated diagnosis by evaluation of VAF. Among the 115 provider- substantiated diagnostic cases with 117 disorders, a causal variant with a VAF below 20% (Supplemental

Table 1) occurred in 5 probands, and contributed to 4.3% [95% CI, 1.4%-9.9%] of our total diagnoses and

7.9% [95% CI, 2.6%-17.6%] of our confirmed de novo variants. As established within our pipeline, heterozygous calls (0/1 genotype) rarely deviate toward extreme allelic proportions, with 0.3% of high- confidence calls within a reference standard occurring at VAF <20 or >80 (Supplemental Fig. 1). In addition, causal variants in 2 probands were found in a mosaic state in 2 unaffected parents (0.3% of available parental samples). All mosaic variants were verified by Sanger sequencing analysis as evidenced by disparate peak height in the electropherogram (Supplemental Fig. 2). In total, mosaic etiology in a proband, or that originating in a parent with transmission to a proband, was associated with 7

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out of 117 (6.0%) provider-substantiated genetic diagnoses. These mosaic variants were recurrently associated with several types of disorders, predominately involving neurodevelopmental features, including early infantile epileptic encephalopathies, intellectual disability syndromes, Coffin-Siris syndrome, and megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome (MPPH) (Table 3).

DISCUSSION

We evaluated the concordance of the molecular ES diagnostic rate generated by the clinical laboratory with ordering clinical provider-substantiated diagnoses. Our results demonstrate the robust clinical utility of ES, with 91.4% of the laboratory findings reported as likely causal for the proband phenotype resulting in a clinically confirmed diagnosis by the ordering provider. This concordance may increase over time, as

4 laboratory-reported diagnostic cases were still under consideration in the patient differential. The high concordance rate may be attributable to the detailed clinical feature information form submitted by an experienced clinical geneticist or genetic counselor at the time of ES ordering, additional curation of the medical record as performed by the variant analysis team or laboratory genetic counselor to enable a phenotype-informed variant analysis approach, as well as the multi-disciplinary expertise achieved by group evaluation of annotated, filtered variants in a case conference setting. Within our primarily pediatric population, we obtained a 32.2% diagnostic yield, with 64.9% of causal variants identified by trio analysis confirmed as de novo. These numbers are in line with previously reported exome and genome sequencing diagnostic yields (average = 31%) and de novo rates (average = 44%) (Clark et al. 2018). In comparison to other large clinical exome sequencing cohorts, our study found a similar rate of cases submitted for trio analysis. In addition, the distribution of AD, AR, and XL inheritance patterns of causal variants were also comparable (Yang et al. 2013; Yang et al. 2014; Lee et al. 2014; Retterer et al. 2016;

Farwell et al. 2015).

Of the 115 diagnostic cases, we confirmed mosaicism of a disease-associated variant in 5 probands and 2 parental samples. The contribution of parental mosaicism may be higher, given that trio analyses were available for 74.8% of our cases. The frequency of mosaicism in our cohort is higher than what has previously been reported in unselected clinical exome sequencing cohorts (Cao et al. 2019;

Yang et al. 2013; Retterer et al. 2016). Several differences between our study and previous reports may

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contribute to this difference, including: sequencing in a hospital-based laboratory representing a pediatric patient population referred by clinical geneticists within a single institution, stringent quality control metrics for sequencing data (including both assay and variant quality measures), diagnostic yield based on EMR clinical substantiation versus solely a laboratory-defined molecular diagnostic yield, and manual evaluation of all assessed variants with specific review of aligned reads and variant characteristics in proband and parental samples.

Our positive mosaic findings contributed to several types of disorders associated with neurodevelopmental features including Coffin-Siris syndrome (n=2), early infantile epileptic encephalopathy (n=2), intellectual disability syndromes (n=2), and the brain overgrowth syndrome MPPH

(n=1). The presence of mosaicism in these patients is consistent with the reported association of mosaic variants within these types of disorders (Stosser et al. 2018; Krupp et al. 2017; Lim et al. 2017; Madsen et al. 2018). The enrichment of our pediatric ES population for various neurodevelopmental features, such as developmental delays, cognitive impairment, and autistic behavior, also may have contributed to the high prevalence of mosaicism seen within this cohort.

Identifying that a disease-causing variant is mosaic can have several implications when counseling families. While the presence of mosaicism is generally unable to predict disease severity, as often only a single tissue type is available for study, it can be informative in providing a clinical explanation for a proband with a mild presentation. In this cohort, detection of mosaicism provided a mechanism to explain a mildly affected male diagnosed with the X-linked disorder, early infantile epileptic encephalopathy 2, primarily seen in females (Case 2). Identifying mosaicism in proband or parental samples is of particular importance to inform recurrence risk in future pregnancies. Evaluating parental samples for the presence of mosaicism can also be beneficial for providing an explanation for why a parent positive for pathogenic variant may be mildly symptomatic or unaffected, as evidenced in this series by the unaffected mother with an IQSEC2 mosaic variant, which was hemizygous in the affected proband, as well as his affected brother (Case 4, previously reported by Barrie et al. 2019).

Mosaic variants can be challenging to detect clinically due to several factors, and consensus guidelines addressing how to detect, assess, and report mosaicism are not currently available. These

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variants can occur at very low frequencies and thus may be below the threshold of detection if coverage

or read depth is insufficient at the affected residue. Acuna-Hidalgo et al. modeled that the probability of

detecting mosaicism at 100x coverage is greater than 90% for variants occurring at 10% VAF or higher.

However, the ability to detect these variants decreases considerably with decreasing read depth (Acuna-

Hidalgo et al. 2015). The higher sequence depth in coding regions typically achieved by exome sequencing thus offers an advantage for detecting mosaicism compared to genome sequencing, due to cost-related limitations to genome sequencing read depth. Despite sufficient coverage across a given region, if the variant is present in a limited number of reads, it may be below the sensitivity of the variant caller software. In our cohort, one of the mosaic variants in a parental sample (Case 6), the ARID2 variant at 4% VAF, was not detected by the variant caller, and came to attention during manual review of reportable variants within aligned sequencing reads. This highlights the utility in manual review of variant calls within the aligned reads to evaluate variant authenticity.

Alternatively, identifying an authentic mosaic variant can also present a challenge. With MPS, false-positive variants may occur by several means. These include biologic contamination during sample acquisition, nucleic acid extraction, or library preparation, low-level admixture during sequencing, or due to sample carryover from prior sequencing runs, as well as PCR or sequencing artifacts (incorporation errors and library chimeras). Additionally, index-hopping can result in false positive calls at very low-level as single-indexed reads can incur mis-assignment of indices within multiplexed libraries, a phenomenon known to be enhanced on patterned flow cells in short-read sequencing chemistries. The confounding variable of index-hopping in the setting of trio analyses can be decreased by the use of dual-indexing of libraries (Costello et al. 2018). To increase confidence in calling mosaicism and rare, low frequency variants, the addition of dual-indexed libraries containing unique identifiers known as molecular barcodes

(Kinde et al. 2011; Schmitt et al. 2012) allows discrimination of improperly indexed libraries and random errors. In this study, all de novo and mosaic variants called with single-indexed libraries were confirmed by Sanger sequencing. Evidence of the low-level variant allele could be visualized for all mosaic variants by review of peak heights in the electropherogram. A priori knowledge of suspected mosaicism facilitated review of Sanger sequencing data and enabled scrutiny of allelic peak heights, including the variant allele relative to background. Thus, data points from two orthogonal methodologies (MPS and Sanger) were

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considered prior to report out of a mosaic call. The variant with a VAF of 4% in a maternal sample (case

6) was visualized by Sanger sequencing, despite being below the threshold typically appreciable by this method (approximately 10-20%), due to the nature of the variant (two nucleotide deletion), as well as prior observation of the heterozygous variant in the proband. However, if the VAF is too low to be appreciable by Sanger, high-depth amplicon sequencing or other quantitative approaches including digital droplet

PCR could be used. The testing of alternative tissue sources can also be considered to aid in confirming suspected mosaicism in patients, though multiple tissue sources may be required, and may be impractical or impossible to obtain. Authentic mosaic variants with high VAFs can also be challenging to detect, as they may be mistaken for heterozygous calls that deviate from the expected 50% VAF due to technical variation and platform bias (Acuna-Hidalgo et al. 2015).

Outside of technical limitations of the assay, mosaic variants can go undetected when the variant is confined to specific tissue types. In these cases, sequencing of the affected tissues is required for detection. Confinement of mosaicism in a parent to predominately or exclusively germline cells would also go undetected by clinical ES trio analysis, and typically only comes to light in instances of multiple affected children from an otherwise unaffected parent.

In conclusion, our study highlights two years of an on-going ES assay within a pediatric tertiary care institution and emphasizes the utility of clinical-provider engagement in ES test ordering and phenotypic curation, as demonstrated by the strong concordance of laboratory-reported and ordering provider-substantiated diagnostic yield. Additionally, we demonstrate that mosaicism is an important contributor to disease-causing variation identified by ES within the pediatric population. Given that our diagnostic yield of mosaic variants exceeds those reported by other ES studies, vigilant manual review of variant calls by a highly skilled variant analysis team and clinical laboratory directors may be a key differentiator in detecting somatic mosaic events. This must occur in concert with adequate read depth and breadth of the assay, appropriate bioinformatic processing parameters, and in consideration of the proband’s clinical characteristics to attribute causality. As we begin to appreciate the expanding role of mosaicism in genetic disease, further research on the types of disorders with mosaicism, clinical implications, and optimal laboratory practices for identifying and reporting mosaicism are needed.

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METHODS

Sequencing, bioinformatics, and quality control

Proband and parental peripheral blood samples were submitted for ES to The Steve and Cindy

Rasmussen Institute for Genomic Medicine at Nationwide Children’s Hospital, Columbus, Ohio. Prior to

ES studies, microarray analysis was previously performed on the submitted proband, unless deemed to not be clinically indicated by the ordering provider (5% of the cohort). Genomic DNA was extracted using the Puregene DNA isolation kit according to the manufacturer protocol (Qiagen, Germantown, MD) or

EZ1 DNA isolation kit (Qiagen), with two separate DNA extractions performed per each submitted proband and parental sample to facilitate identity and provenance analyses. Genotyping of 30 autosomal loci representing single nucleotide variants with high population minor allele frequency was performed in the parental and proband samples using a custom Agena MassArray panel (Agena, San Diego, CA).

Subsequently, a comparison of the Agena-derived genotype data was performed relative to aligned sequencing read data derived by MPS to further ensure sample provenance throughout the extraction, library preparation, sequencing, and bioinformatic analysis stages, as well as to verify familial relationships.

Libraries were subject to target capture using SureSelect Human All Exon V6 (Agilent, Santa

Clara, CA) followed by paired-end 101 or 151bp sequencing to 137x mean depth on a HiSeq 2500 or

HiSeq 4000 (Illumina, San Diego, CA), with 96.5% of targeted bases at 20X or greater (Supplemental

Table 2). Sequencing data were demultiplexed and analyzed by GenomeNext (Columbus, OH) v1.1, which performs alignment to the reference sequence (GRCh37/hg19 Feb 2009), deduplication, and single sample variant calling with GATK Unified Genotyper 1.6-13 via the Churchill secondary analysis pipeline

(Kelly et al. 2015). Mitogen (Sunquest, Tucson, AZ) was used for annotation and tertiary analysis filtering informed by clinician-provided phenotypes, which were converted into Human Phenotype Ontology (HPO) terms (Kohler et al. 2017).

As standard of practice in our exome workflow, aligned sequencing reads in the BAM file were reviewed at all assessed variants using the Integrative Genomics Viewer v2.3-2.4.4 (Broad) (Robinson et

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al. 2011). This allowed for a review of variant authenticity and encompassed an examination of read

counts and strandedness, location of the variant within the read, VAF, homology, read quality and

mapping. Manual review of read-aligned proband data was performed relative to review of identity-

confirmed parental sequence to allow for side-by-side comparison, with IGV alignment preferences set to

allow for display of coverage allele-fraction threshold at 2% and retention of soft-clipped reads. Genomic regions with known homology as defined by Mandelker et al. (2016) have been incorporated into ES analysis through BED file track definitions to visually flag the level of homology of the local region in IGV.

Parentage was confirmed in our dataset for all de novo calls, with familial relationships established by genotyping per our standard workflow. Hemizygous variants were X-linked variants identified in >95% of reads in male patients. Homozygous calls applied to autosomal variants in >95% of reads. Variants deviating to the extreme of VAF <20 or >80 were considered suspicious for mosaicism, and subsequently underwent orthogonal testing by Sanger sequencing.

Sanger sequencing was performed on proband and available parental samples for all de novo

variants and suspected mosaic variants identified as likely causal for the proband phenotype. Analysis by

Sanger was used to distinguish from MPS or PCR artifact, and verify reduction in allelic ratio as visualized

by disparate peak heights for mosaicism. For Sanger sequencing, PCR amplification of the region of

interest was followed by purification using the QIAquick purification kit (Qiagen, Germantown, MD).

Forward and reverse sequencing reactions were performed with the Big Dye v3.1 terminator mix

(ThermoFisher, Waltham, MA). Sequencing was performed on an Applied Biosystems 3130 or 3730

instrument (ThermoFisher, Waltham, MA). Mosaic variants were confirmed by observation of a non-

reference allele with a disparate peak height during Sanger analysis.

Variant interpretation

Custom scripting allowed for enrichment of variant attribute data including disease association and

phenotype overlap. For each proband, the annotated filtered variant list was evaluated at a case conference attended by laboratory directors, genetic counselors, variant scientists, residents, fellows, and geneticists, including the ordering provider, when available. Variants that met group consensus were assessed according to ACMG/AMP recommendations (Richards et al. 2015). Following assessment,

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variants are reported as either likely causal for proband phenotype or as findings of undetermined clinical relevance to the proband phenotype based upon strength of phenotype overlap with the associated

disease at the discretion of the ABMGG board-certified signing director. Laboratory diagnostic yield was

defined as the number of cases with a variant or variants reported as likely causal for the proband

phenotype. Concordance between the laboratory and the ordering provider as to reported variant-

associated disease causality was examined via clinical documentation in the EMR. Variants were

considered clinically confirmed as causal if the ordering provider attributed some or all features in the

proband to the variant(s).

ADDITIONAL INFORMATION

Data Deposition and Access

The mosaic variants ClinVar (http://www.ncbi.nlm.nih.gov/clinvar/) IDs are SCV001161761.1,

SCV001161762.1, SCV001161763.1, SCV000864353.2, SCV001161764.1, SCV001161765.1, and

SCV001161766.1. Additional variants from the 117 diagnostic cases have been deposited to ClinVar

under submitter: Institute for Genomic Medicine (IGM) Clinical Laboratory, Nationwide Children's Hospital.

Details are provided in the supplemental material. Deposition of raw sequencing data is not permitted

based on patient consent.

Ethics Statement

All patients or their guardians provided written informed consent for genomic sequencing. This research is

under a protocol approved by the Institutional Review Board at Nationwide Children’s Hospital (IRB18-

00662).

Acknowledgments

We thank the patients, families, and clinicians involved in these cases. We acknowledge Maria Alfaro,

PhD, Erik Zmuda, PhD, and Julie Gastier-Foster, PhD for their contributions in clinical patient care.

Author Contributions

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CRM prepared the manuscript. All authors contributed to design of workflows and data procurement, and reviewed and approved the manuscript. CEC supervised the study.

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Table 1. Frequency of the top 20 HPO terms used to describe features of 357 probands referred for exome sequencing

Number of HPO ID HPO term probands (%)

HP:0001263 Global developmental delay 262 (73.4)

HP:0001999 Abnormal facial shape 183 (51.3)

HP:0001252 Muscular hypotonia 182 (51.0)

HP:0000750 Delayed speech and language development 157 (44.0)

HP:0001270 Motor delay 136 (38.1)

HP:0002194 Delayed gross motor development 127 (35.6)

HP:0001250 Seizures 119 (33.3)

HP:0100543 Cognitive impairment 98 (27.5)

HP:0001508 Failure to thrive 91 (25.5)

HP:0004322 Short stature 90 (25.2)

HP:0007010 Poor fine motor coordination 77 (21.6)

HP:0000252 Microcephaly 74 (20.7)

HP:0002020 Gastroesophageal reflux 69 (19.3)

HP:0000729 Autistic behavior 69 (19.3)

HP:0000717 Autism 59 (16.5)

HP:0001290 Generalized hypotonia 57 (16.0)

HP:0001622 Premature birth 56 (15.7)

HP:0002019 Constipation 56 (15.7)

HP:0001510 Growth delay 52 (14.6)

HP:0002376 Developmental regression 50 (14.0)

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Table 2. Summary of cases with a pathogenic or likely pathogenic mosaic variant in a proband or parental sample

Variant Parent Relevant Proband type/ of Zygosity Chromosome HGVS DNA and disease Case Gender/ Clinical phenotype Gene Predicted origin/ Supporting references /VAF (hg19) protein reference association Age effect VAF

Failure to thrive, profound hypotonia, global developmental delay, NM_139058.2 Male/ microcephalic, bilateral esotropia, Substitution/ (XL) Early Shoubridge et al. 2012 short palpebral fissures, protuberant Mosaic chrX:25025232 c.1444G>A infantile epileptic Shoubridge et al. 2010 1 6 mo ARX Missense de novo tongue, sparse scalp hair, (12%) C>T p.(Gly482Ser) encephalopathy Gronskov et al. 2014 hypsarrhythmia by long-term 1 Poirier et al. 2005 electroencephalographic monitoring, delayed myelination on MRI

Bahi-Buisson et al. 2012 Generalized epilepsy, global Kilstrup-Nielsen et al. 2012 developmental delay, intellectual (XL) Early NM_003159.2 Mei et al. 2014 Male/ disability, autism, attention deficit Mosaic chrX:18602452 Substitution/ infantile epileptic 2 CDKL5 c.533G>A de novo Masliah-Plachon et al. 2010 11 yo hyperactivity disorder, able to walk (17%) G>A Missense encephalopathy p.(Arg178Gln) Stosser et al. 2018 independently with orthotics and 2 Kothur et al. 2018 verbally communicate Olson et al. 2019

Global developmental delay, (AD) Clark- Bramswig et al. 2017 Male/ seizures, chorea, hypotonia, short Mosaic chr2:230679862 NM_004238.2 Substitution/ 3 TRIP12 de novo Baraitser Zhang et al. 2017 1 yo stature, poor feeding, ptosis, frontal (12%) G>A c.1540C>T Nonsense syndrome Louie et al., 2020 bossing, micrognathia p.(Arg514Ter)

Localization-related partial epilepsy with complex partial seizures, non- Barrie et al. 2019* ambulatory, global developmental NM_001111125.2 mosaic Male/ Hemi chrX:53264051 Substitution/ (XL) Mental Ewans et al. 2017 4 delay, postnatal growth retardation, IQSEC2 c.3817C>T mother 7 yo (98%) G>A Nonsense retardation 1 Radley et al. 2019 intellectual disability, autism, p.(Gln1273Ter) (11%) Mignot et al. 2019 hyperopia, chronic constipation, dysphagia

Global developmental delay, fine motor delay, hirsutism, agenesis of NM_139135.2 Tsurusaki et al. 2012 Female/ the corpus callosum, hypotonia, Mosaic chr1:27092947 Substitution/ (AD) Coffin-Siris 5 ARID1A c.2879-1G>A de novo Santen et al. 2013 8 yo tethered spinal cord, exotropia, club (19%) G>A Splicing syndrome 2 N/A Wieczorek et al. 2013 foot, tall forehead, downslanting palpebral fissures, macrostomia

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Periventricular white matter changes on MRI, mild intellectual disability, NM_152641.3 Tsurusaki et al. 2012 chr12: mosaic Male/ global developmental delay, Het c.3927_3928delAG Deletion/ (AD) Coffin-Siris Bramswig et al. 2017 6 ARID2 46245833_4624 mother 7 yo hypotonia, GERD, myopathic facies, (46%) p.(Gly1310Glufs Frameshift syndrome 6 Bogershausen and Wollnik 5834delAG (4%) thickened low-set ears, flared nasal Ter5) 2018 alae, upturned nasal tip (AD) Macrocephaly, hirsutism, global Megalencephaly- NM_005027.3 Mirzaa et al. 2015 Female/ developmental delay, bilateral Mosaic chr19:18273784 Substitution/ polymicrogyria- 7 PIK3R2 c.1117G>A de novo Riviere et al. 2012 2 yo perisylvian polymicrogyria with mildly (18%) G>A Missense polydactyly- p.(Gly373Arg) Madsen et al. 2018 enlarged ventricles on MRI hydrocephalus syndrome 1

Abbreviations: mo, months old; yo, years old; VAF, variant allele frequency/fraction; hemi, hemizygous; het, heterozygous; N/A, not applicable; XL, X-linked; AD, autosomal dominant; * case previously reported

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Table 3: Summary of causal variants in a pediatric exome cohort

Case Study Gene Chromosome HGVS DNA and protein Zygosity Parent Relevant disease association Variant ACMG/AMP criteria type (hg19) reference of origin assessment met AUTOSOMAL RECESSIVE (Homozygous) 8* trio AMPD2 chr1:110172910 NM_001257360.1 Hom Mat/Pat (AR) Pontocerebellar hypoplasia, type 9 VUS PM2, PP3 C>T c.2201C>T (OMIM: 615809) p.(Pro734Leu) (AR) ?Spastic paraplegia 63 (OMIM: 615686)

COL11A1 chr1:103488375 NM_080629.2 Hom Mat/Pat (AD) Stickler syndrome, type II Likely PVS1, PM2 C>A c.1204G>T (OMIM: 604841) Pathogenic p.(Glu402Ter) (AR) Fibrochondrogenesis 1 (OMIM: 228520) (AD) Marshall syndrome (OMIM: 154780)

9 trio AP4S1 chr14:31542174 NM_001254727.1 Hom Mat/Pat (AR) Spastic paraplegia 52, autosomal Pathogenic PVS1, PM2, PM3, C>T c.289C>T recessive (OMIM: 614067) PP1 p.(Arg97Ter)

10 trio HYLS1 chr11:125769895 NM_145014.2 Hom Mat/Pat (AR) Hydrolethalus syndrome Pathogenic PS3, PM1, A>G c.632A>G (OMIM: 236680) PS4_Moderate, PP1, p.(Asp211Gly) PP3, PP5

11 trio IDUA chr4:996535 NM_000203.4 Hom Mat/Pat (AR) Mucopolysaccharidosis Ih Pathogenic PVS1, PS3, G>A c.1205G>A (OMIM: 607014) PS4_Moderate, PP5 p.(Trp402Ter) (AR) Mucopolysaccharidosis Ih/s (OMIM: 607015) (AR) Mucopolysaccharidosis Is (OMIM: 607016)

12* trio KERA chr12:91449224 NM_007035.3 Hom Mat/Pat (AR) Cornea plana 2, autosomal Pathogenic PVS1, PM2, G>A c.835C>T recessive (OMIM: 217300) PS4_Moderate, PP1 p.(Arg279Ter)

POLR3B chr12:106850924 NM_018082.5 Hom Mat/Pat (AR) Leukodystrophy, hypomyelinating, Likely PM2, PM5, PP2, PP3 C>T c.2302C>T 8, with or without oligodontia and/or Pathogenic p.(Arg768Cys) hypogonadotropic hypogonadism (OMIM: 614381) 13 trio PARN chr16:14704607 NM_002582.3 Hom Mat/Pat (AR) Dyskeratosis congenita, autosomal Likely PM1, PM2, G>A c.448C>T recessive 6 (OMIM: 616353) Pathogenic PS3_Moderate, PP3 p.(Arg150Cys)

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14 singleton PYCR1 chr17:79892603 NM_001282281.1 Hom Unknown (AR) Cutis laxa, autosomal recessive, VUS PM1, PM2, PP3 C>T c.640G>A type IIB (OMIM: 612940) p.(Ala214Thr) (AR) Cutis laxa, autosomal recessive, type IIIB (OMIM: 614438)

AUTOSOMAL RECESSIVE (Compound heterozygous)

15 trio AP4M1 chr7:99704117 NM_004722.3 Het Pat (AR) Spastic paraplegia 50, autosomal Pathogenic PVS1, PM2, PM3 C>T c.1117C>T recessive (OMIM: 612936) p.(Gln373Ter)

AP4M1 chr7:99704464 NM_004722.3 Het Mat (AR) Spastic paraplegia 50, autosomal Likely PM2, PM3, PP3, PP5 C>T c.1321C>T recessive (OMIM: 612936) Pathogenic p.(Arg441Ter)

16 singleton HBB chr11:5248232 NM_000518.4 Het Unknown (AR) Sickle cell anemia (OMIM: 603903) Pathogenic PS3, PM3, PM5, T>A c.20A>T PS4_Moderate, PP5 p.(Glu7Val)

HBB chr11:5246908 NM_000518.4 Het Unknown (AR) Sickle cell anemia (OMIM: 603903) Likely PS4, PM2, PM3, PP5 C>T c.364G>A Pathogenic p.(Glu122Lys)

17 trio MED25 chr19:50331716 NM_030973.3 Het Pat (AR) ?Charcot-Marie-Tooth disease, type Likely PM2, PM3, PP2, PM5 G>A c.316G>A 2B2 (OMIM: 605589) Pathogenic p.(Gly106Arg) (AR) Basel-Vanagait-Smirin-Yosef syndrome (OMIM: 616449)

MED25 chr19:50338388_5 NM_030973.3 Het Mat (AR) ?Charcot-Marie-Tooth disease, type Likely PVS1, PM2 0338397delACCAC c.1628_1637delACCAC 2B2 (OMIM: 605589) Pathogenic AAGCA AAGCA (AR) Basel-Vanagait-Smirin-Yosef p.(Asn543ArgfsTer51) syndrome (OMIM: 616449)

18 duo OPA1 chr3:193374977 NM_130837.2 Het Unknown (AR) Behr syndrome (OMIM: 210000) Pathogenic PVS1, PM2 delA c.2287delA (AR) Optic atrophy 1 (OMIM: 165500) p.(Ser763ValfsTer15) (AR) Optic atrophy plus syndrome (OMIM: 125250)

OPA1 chr3:193361167 NM_130837.2 Het Mat (AR) Behr syndrome (OMIM: 210000) VUS PM1, PM3, PP1, PP3; A>G c.1311A>G (AR) Optic atrophy 1 (OMIM: 165500) BS3 p.(Ile437Met) (AR) Optic atrophy plus syndrome (OMIM: 125250)

19 trio RYR1 chr19:38951159 NM_000540.2 Het Pat (AR) Minicore myopathy with external Pathogenic PVS1, PM2, PP5 delG c.2505delG ophthalmoplegia (OMIM: 255320) p.(Pro836LeufsTer48) (AD, AR) Central core disease (OMIM: 117000)

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20 trio RYR1 chr19:38987047 NM_000540.2 Het Pat (AR) Minicore myopathy with external Pathogenic PVS1, PM2, PP3 A>G c.6664-2A>G ophthalmoplegia (OMIM: 255320) p.? (AD, AR) Central core disease (OMIM: 117000)

RYR1 chr19:g.39075637 NM_000540.2 Het Mat (AR) Minicore myopathy with external Likely PM1, PM3, PP2, PP3 G>A c.14701G>A ophthalmoplegia (OMIM: 255320) Pathogenic p.(Glu4901Lys) (AD, AR) Central core disease (OMIM: 117000) 21 trio SAMHD1 chr20:35563513 NM_015474.3 Het Mat (AR) Aicardi-Goutieres syndrome 5 Pathogenic PS3, PM2, PM5, PP3, C>T c.428G>A (OMIM: 612952) PP5 p.(Arg143His)

SAMHD1 chr20:35533834 NM_015474.3 Het Pat (AR) Aicardi-Goutieres syndrome 5 Likely PM2, PM3, PP3, PP4 A>G c.1343T>C (OMIM: 612952) Pathogenic p.(Ile448Thr) 22 duo SLC17A5 chr6:74351533 NM_012434.4 Het Unknown (AR) Salla disease (OMIM: 604369) Pathogenic PS3, PM2, T>C c.406A>G (AR) Sialic acid storage disorder, PS4_Moderate, PP3, p.(Lys136Glu) infantile (OMIM: 269920) PP5

SLC17A5 chr6:74345115 NM_012434.4 Het Mat (AR) Salla disease (OMIM: 604369) Pathogenic PVS1, PM2, PM3 A>T c.809T>A (AR) Sialic acid storage disorder, p.(Leu270Ter) infantile (OMIM: 269920) 23 trio SLC22A5 chr5:131706028 NM_003060.3 Het Mat (AR) Carnitine deficiency, systemic Likely PS3, PM2, PM3, PP3 G>T c.364G>T primary (OMIM: 212140) Pathogenic p.(Asp122Tyr)

SLC22A5 chr5:131721062 NM_003060.3 Het Pat (AR) Carnitine deficiency, systemic Pathogenic PS3, PM_PS4, PM2, C>T c.695C>T primary (OMIM: 212140) PP3, PP5 p.(Thr232Met)

24 trio SLC6A3 chr5:1422127 NM_001044.4 Het Mat (AR) Parkinsonism-dystonia, infantile, 1 VUS PM2, PP2, PP3 C>T c.656G>A (OMIM: 613135) p.(Arg219His)

SLC6A3 chr5:1416253 NM_001044.4 Het Pat (AR) Parkinsonism-dystonia, infantile, 1 VUS PM2, PP2, PP3 C>G c.991G>C (OMIM: 613135) p.(Ala331Pro) 25 trio SRD5A2 chr2:31758741 NM_000348.3 Het Mat (AR) Pseudovaginal perineoscrotal Pathogenic PS3, PM2, PM3, T>C c.377A>G hypospadias (OMIM: 264600) PS4_Moderate, PP1, p.(Gln126Arg) PP5

SRD5A2 chr2:31758802 NM_000348.3 Het Pat (AR) Pseudovaginal perineoscrotal Likely PVS1, PM2 delG c.317delC hypospadias (OMIM: 264600) Pathogenic p.(Pro106LeufsTer25)

26 trio TBCD chr17:80772747 NM_005993.4 Het Mat (AR) Encephalopathy, progressive, early- VUS PM2, PP3 G>A c.1255G>A onset, with brain atrophy and thin corpus p.(Gly419Arg) callosum (OMIM: 617193)

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TBCD chr17:80882859_8 NM_005993.4 Het Pat (AR) Encephalopathy, progressive, early- VUS PM2 0882861delGAG c.2305_2307delGAG onset, with brain atrophy and thin corpus p.(Glu769del) callosum (OMIM: 617193)

27 trio TRNT1 chr3:3189583 NM_182916.2 Het Pat (AR) Retinitis pigmentosa and Likely PM2, PM3, PP3, PP5 C>G c.1057-7C>G erythrocytic microcytosis Pathogenic p.? (OMIM: 616959) (AR) Sideroblastic anemia with B-cell immunodeficiency, periodic fevers, and developmental delay (OMIM: 616084)

TRNT1 chr3:3189779 NM_182916.2 Het Mat (AR) Retinitis pigmentosa and Likely PM2, PM3, PM_PS3 A>G c.1246A>G erythrocytic microcytosis Pathogenic p.(Lys416Glu) (OMIM: 616959) (AR) Sideroblastic anemia with B-cell immunodeficiency, periodic fevers, and developmental delay (OMIM: 616084) 28 trio UBA5 chr3:132379541 NM_024818.3 Het Mat (AR) Epileptic encephalopathy, early Pathogenic PVS1, PM1, dupA c.160dupA infantile, 44 (OMIM: 617132) PM2,PP3 p.(Ser54LysfsTer16)

UBA5 chr3:132384835 NM_024818.3 Het Pat (AR) Epileptic encephalopathy, early Likely PM1, PM2, PM3, PP2 G>A c.215G>A infantile, 44 (OMIM: 617132) Pathogenic p.(Arg72His)

AUTOSOMAL RECESSIVE (One SNV + one copy loss)

29 trio IFT140 chr16:1642177 NM_014714.3 Hemi Mat (AR) Short-rib thoracic dysplasia 9 with Pathogenic PVS1, PS3, PM2, C>T c.634G>A (suspected or without polydactyly (OMIM: 266920) PM3, PP3, PP5 p.(Gly212Arg) deletion on (AR) Retinitis pigmentosa 80 other allele) (OMIM: 617781)

30 trio NUBPL chr14:32319298 NM_025152.2 Hemi Mat (AR) Mitochondrial complex I deficiency, Likely PS3, PP3, PP5; BS1 T>C c.815-27T>C (~400kb nuclear type 21 (OMIM: 618242) Pathogenic p.? deletion on other allele, by array)

AUTOSOMAL DOMINANT 31 trio ABCC9 chr12:21995260 NM_020297.3 Het De novo (AD) Hypertrichotic Pathogenic PS1, PS2, PS3 PM2, C>T c.3461G>A osteochondrodysplasia (OMIM: 239850) PP2, PP3 p.(Arg1154Gln)

32 trio ANKRD11 chr16:89345988_8 NM_001256183.1 Het De novo (AD) KBG syndrome (OMIM: 148050) Pathogenic PVS1, PS2, PM2 9345989insGGCTT c.6968_6975dupCCCC CGG GAAG p.(Ala2326ProfsTer14)

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33 trio ANKRD11 chr16:89349005 NM_001256182.1 Het De novo (AD) KBG syndrome (OMIM: 148050) Pathogenic PVS1, PS2, PM2, delC c.3948delG PM4 p.(Leu1317Ter) 34 trio ANKRD11 chr16:89351049_8 NM_001256182.1 Het De novo (AD) KBG syndrome (OMIM: 148050) Pathogenic PVS1, PS2, PM2, 9351053delGTTTT c.1903_1907delAAACA PS4_Moderate, PP1, p.(Lys635GlnfsTer26) PP5

35 singleton ANKRD11 chr16:89351049_8 NM_001256182.1 Het Unknown (AD) KBG syndrome (OMIM: 148050) Pathogenic PVS1, PS2, PM2, 9351053delGTTTT c.1903_1907delAAACA PS4_Moderate, PP1, p.(Lys635GlnfsTer26) PP5

36 trio ABCC9 chr12:21995375 NM_005691.3 Het Mat (AD) Hypertrichotic Likely PM2, PM5, PP2, PP3, G>A c.3346C>T osteochondrodysplasia (OMIM: 610253) Pathogenic PP5 p.(Arg1116Cys) 37 trio ACTB chr7:5568223 NM_001101.3 Het De novo (AD) ?Dystonia, juvenile-onset Likely PS2, PM2, PP2, PP3 G>T c.491C>A (OMIM: 607371) Pathogenic p.(Pro164His) (AD) Baraitser-Winter syndrome 1 (OMIM: 243310) 38 trio ARID1B chr6:157528657 NM_020732.3 Het De novo (AD) Coffin-Siris syndrome 1 Pathogenic PVS1, PS2, PM2, C>T c.6382C>T (OMIM: 135900) PP5 p.(Arg2128Ter) 39 trio ATP6V1A chr3:113508666 NM_001690.3 Het De novo (AD) Epileptic encephalopathy, infantile Pathogenic PS2, PM1, PM2, PP2, G>A c.967A>G or early childhood, 3 (OMIM: 618012) PP3 p.(Arg323Gly) 40 trio CACNA1C chr12:2566837 NM_199460.3 Het De novo (AD) Timothy syndrome (OMIM: 601005) Likely PS2, PM1, PM2, PP3 T>C c.722T>C (AD) Brugada syndrome 3 Pathogenic p.(Val241Ala) (OMIM: 611875)

41 trio CAMTA1 chr1:7798409_779 NM_015215.3 Het Mat (AD) Cerebellar ataxia, nonprogressive, Pathogenic PVS1, PM2, PP1 8411delinsGTGCT c.4049_4051delinsGTG with mental retardation (OMIM: 614756) GC CTGC p.(Pro1350ArgfsTer18)

42 trio CAV3 chr3:8787396 NM_001234.4 Het De novo (AD) Cardiomyopathy, familial Likely PS2, PM1, PM2, PP3 T>A c.299T>A hypertrophic (OMIM: 192600) Pathogenic p.(Ile100Asn) (AD) Creatine phosphokinase, elevated serum (OMIM: 123320) (AD) Long QT syndrome 9 (OMIM: 611818) (AD) Myopathy, distal, Tateyama type (OMIM: 614321) (AD) Rippling muscle disease (OMIM: 606072) 43 trio CCND2 chr12:4409144 NM_001759.3 Het De novo (AD) Megalencephaly-polymicrogyria- Pathogenic PS2, PM1, PM2, C>A c.839C>A polydactyly-hydrocephalus syndrome 3 PS4_Moderate, PP5 p.(Thr280Asn) (OMIM: 615938)

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44 duo CHD2 chr15:93485148 NM_001271.3 Het Unknown (AD) Epileptic encephalopathy, Likely PVS1, PM2 dupT c.789dupT childhood-onset (OMIM: 615369) Pathogenic p.(Glu264Ter)

45 trio CHD7 chr8:61773555_61 NM_017780.3 Het De novo (AD) CHARGE syndrome Pathogenic PVS1, PS2, PM2 773556 c.7701_7702delAC (OMIM: 214800) delAC p.(Arg2568AspfsTer7) (AD) Hypogonadotropic hypogonadism 5 with or without anosmia (OMIM: 612370)

46 singleton COL3A1 chr2:189856222 NM_000090.3 Het Unknown (AD) Ehlers-Danlos syndrome, vascular Likely PM1, PM2, PP2, PP3 G>T c.862G>T type (OMIM: 130050) Pathogenic p.(Gly288Cys)

47 trio CREBBP chr16:3779691 NM_004380.2 Het De novo (AD) Rubinstein-Taybi syndrome 1 Likely PS2, PM1, PM2, PP3 C>T c.5357G>A (OMIM: 180849) Pathogenic p.(Arg1786His) 48 trio CREBBP chr16:3789597 NM_004380.2 Het De novo (AD) Rubinstein-Taybi syndrome 1 Likely PS2, PM1, PM2, PP3 C>A c.4262G>T (OMIM: 180849) Pathogenic p.(Cys1421Phe) 49 trio CREBBP chr16:3842042 NM_004380.2 Het De novo (AD) Rubinstein-Taybi syndrome 1 Pathogenic PVS1, PS2, PM2, G>A c.1270C>T (OMIM: 180849) PP5 p.(Arg424Ter) 50 trio CSNK2A1 chr20:476405 NM_177559.2 Het De novo (AD) Okur-Chung neurodevelopmental Pathogenic PS2, PM1, PM2, A>T c.468T>A syndrome (OMIM: 617062) PM5, PP2, PP3, PP5 p.(Asp156Glu) 51 trio CSNK2A1 chr20:485826 NM_177559.2 Het De novo (AD) Okur-Chung neurodevelopmental Pathogenic PS2, PM2, PM5, PP2, T>C c.149A>G syndrome (OMIM: 617062) PP5 p.(Tyr50Cys) 52 trio DYRK1A chr21:38862468_3 NM_101395.2 Het De novo (AD) Mental retardation, autosomal Likely PS2, PM2, PP5 8862472 c.665-9_665- dominant 7 (OMIM: 614104) Pathogenic delCTCTT 5delCTCTT p.?

53 singleton EFTUD2 chr17:42964119 NM_004247.3 Het Unknown (AD) Mandibulofacial dysostosis, Guion- Pathogenic PVS1, PM2, PP3 C>G c.106-1G>C Almeida type (OMIM: 610536) p.?

54 duo EHMT1 chr9:140637869 NM_024757.4 Het Unknown (AD) Kleefstra syndrome 1 Likely PVS1, PM2 dupA c.870dupA (OMIM: 610253) Pathogenic p.(Arg291ThrfsTer7) 55 trio EHMT1 chr9:140672394 NM_024757.4 Het De novo (AD) Kleefstra syndrome 1 Pathogenic PVS1, PS2, PM2 dupC c.2079dupC (OMIM: 610253) p.(Glu694ArgfsTer4) 56 trio FLG** chr1:152285861 NM_002016.1 Het Mat (AD) Ichthyosis vulgaris (OMIM: 146700) Pathogenic PVS1, PM3, PP5 G>A c.1501C>T (AD) {Dermatitis, atopic, susceptibility to, p.(Arg501Ter) 2} (OMIM: 605803)

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FLG** chr1:152285081_1 NM_002016.1 Het Pat (AD) Ichthyosis vulgaris (OMIM: 146700) Pathogenic PVS1, PS4, PM3, 52285084delACGT c.2282_2285delCAGT (AD) {Dermatitis, atopic, susceptibility to, PP1 p.(Ser761CysfsTer36) 2} (OMIM: 605803)

57 trio FLG** chr1:152285861 NM_002016.1 Het Pat (AD) Ichthyosis vulgaris (OMIM: 146700) Pathogenic PVS1, PS3, PP1, PP5 G>A c.1501C>T (AD) {Dermatitis, atopic, susceptibility to, p.(Arg501Ter) 2} (OMIM: 605803) 58 trio FOXG1 chr14:29237138 NM_005249.4 Het De novo (AD) Rett syndrome, congenital variant Likely PS2, PM2, PP3 A>G c.653A>G (OMIM: 613454) Pathogenic p.(Tyr218Cys) 59 trio FOXP1 chr3:71026799_71 NM_001244810.1 Het De novo (AD) Mental retardation with language Pathogenic PVS1, PS2, PM2 026802delTAAT c.1420_1423delATTA impairment and with or without autistic p.(Ile474GlyfsTer12) features (OMIM: 613670)

60 duo GABRB2 chr5:160758119 NM_021911.2 Het Unknown (AD) Epileptic encephalopathy, infantile VUS PM1, PM2, PP3 A>G c.848T>C or early childhood, 2 (OMIM: 617829) p.(Leu283Pro)

61 trio GABRB3 chr15:27017618 NM_000814.5 Het De novo (AD) Epileptic encephalopathy, early Likely PS2, PM2, PP3 T>A c.173-2A>T infantile, 43 (OMIM: 617113) Pathogenic p.?

62 trio GABRG2 chr5:161569244 NM_198903.2 Het De novo (AD) Epilepsy, generalized, with febrile Pathogenic PS2, PS3, PM2, PM5, C>T c.964C>T seizures plus, type 3 (OMIM: 607681) PP2, PP3, PP5 p.(Pro322Ser) (AD) Epileptic encephalopathy, early infantile, 74 (OMIM: 618396)

63 singleton HNRNPK chr9:86586597 NM_031263.3 Het Unknown (AD) Au-Kline syndrome Pathogenic PVS1, PS2, PM2, dupT c.998dupA (OMIM: 616580) PP5 p.(Tyr333Ter)

64 trio HRAS chr11:534289 NM_005343.2 Het De novo (AD) Costello syndrome (OMIM: 218040) Pathogenic PS2, PM1, PM2, C>T c.34G>A (AD) Congenital myopathy with excess of PM5, PP5 p.(Gly12Ser) muscle spindles (OMIM: 218040)

65 trio KANSL1 chr17:44248468 NM_001193465.1 Het De novo (AD) Koolen-De Vries syndrome Pathogenic PVS1, PS2, PM2, G>A c.1042C>T (OMIM: 610443) PP5 p.(Arg348Ter) 66 singleton KMT2D chr12:49436599 NM_003482.3 Het Unknown (AD) Kabuki syndrome 1 Pathogenic PVS1, PM2, PM6, G>A c.5707C>T (OMIM: 147920) PP3, PP5 p.(Arg1903Ter) 67 trio MED13L chr12:116452999_ NM_015335.4 Het De novo (AD) Mental retardation and distinctive Pathogenic PVS1, PS2, PM2 116453015 c.1077_1093del facial features with or without cardiac delTCTTTGGACT p.(Met359IlefsTer38) defects (OMIM: 616789) GTGCATC (AD) Transposition of the great arteries, dextro-looped 1 (OMIM: 608808)

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68 trio MYT1L chr2:1915819 NM_015025.3 Het De novo (AD) Mental retardation, autosomal Pathogenic PS2, PM1, PM2, PP3, A>T c.1676T>A dominant 39 (OMIM: 616521) PP2 p.(Val559Asp)

69 trio MYT1L chr2:1906916 NM_001303052.1 Het De novo (AD) Mental retardation, autosomal Pathogenic PVS1, PS2, PM2 A>T c.1968T>A dominant 39 (OMIM: 616521) p.(Tyr656Ter)

70 trio NBEA chr13:35923346_3 NM_015678.4 Het De novo (AD) Seizures & intellectual disability (No VUS PM2, PS2_Moderate, 5923347delAG c.6005_6006delAG OMIM) (PMID:28554332) PP3 p.(Glu2002ValfsTer2) 71 trio NEDD4L chr18:56033460 NM_001144967.2 Het Mat (AD) Periventricular nodular heterotopia VUS PM1, PM2, PP3 C>G c.2063C>G 7 (OMIM: 617201) p.(Thr688Arg) 72 trio NOTCH1 chr9:139399384_1 NM_017617.3 Het Pat (AD) Adams-Oliver syndrome 5 Likely PVS1, PM2 39399385insTG c.4758_4759insCA (OMIM: 616028) Pathogenic p.(Asn1587GlnfsTer30) (AD) Aortic valve disease 1 (OMIM: 109730) 73 trio NSD2 chr4:1906053 NM_133330.2 Het De novo (AD) Wolf-Hirchhorn syndrome-like Pathogenic PVS1, PS2, PM2, G>A c.708G>A (PMID: 31171569, 29884796) PP3 p.(Trp236X) 74 trio NSD2 chr4:1936884dupG NM_133330.2 Het De novo (AD) Wolf-Hirchhorn syndrome-like Pathogenic PVS1, PS2, PM2 c.1569dupG (PMID: 31171569, 29884796) p.(Lys524GlufsX17) 75 trio NSD2 chr4:1918630 NM_133330.2 Het De novo (AD) Wolf-Hirchhorn syndrome-like Pathogenic PVS1, PS2, PM2 C>T c.793C>T (PMID: 31171569, 29884796) p.(Gln265Ter) 76 trio PPP2R5D chr6:42975698 NM_006245.3 Het De novo (AD) Mental retardation, autosomal Pathogenic PS2, PM2, PM5, PP2, A>C c.752A>C dominant 35 (OMIM: 616355) PP3 p.(Asp251Ala) 77 singleton PTPN11 chr:12:112888172 NM_002834.3 Het Unknown (AD) Noonan syndrome 1 Pathogenic PS1, PS3, PS4, A>G c.188A>G (OMIM: 163950) PP1_Strong, PM1, p.(Tyr63Cys) (AD) LEOPARD syndrome 1 PP2, PP3 (OMIM: 151100) (AD) Metachondromatosis (OMIM: 156250) 78 trio PTPN11 chr12:112915523 NM_002834.3 Het De novo (AD) Noonan syndrome 1 Pathogenic PS2_VERYStrong, A>G c.922A>G (OMIM: 163950) PS3, PS4, p.(Asn308Asp) (AD) LEOPARD syndrome 1 PP1_Strong, PM1, (OMIM: 151100) PP2, PP3 (AD) Metachondromatosis (OMIM: 156250) 79 trio PUF60 chr8:144898801 NM_078480.2 Het De novo (AD) Verheij syndrome (OMIM: 615583) Pathogenic PVS1, PS2, PM2 dupA c.1569dupT p.(Glu524Ter)

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80 singleton RAI1 chr17:17700943 NM_030665.3 Het Unknown (AD) Smith-Magenis syndrome Pathogenic PVS1, PM2, PP3 C>T c.4681C>T (OMIM: 182290) p.(Arg1561Ter) 81 trio SALL1 chr16:51174260 NM_002968.2 Het Pat (AD) Townes-Brocks syndrome 1 Pathogenic PVS1, PM2, PP1, C>A c.1873G>T (OMIM: 107480) PP3 p.(Glu625Ter) 82 trio SATB2 chr2:200193509 NM_015265.3 Het De novo (AD) Glass syndrome (OMIM: 612313) Likely PS2, PM1, PM2, PP2 T>G c.1298A>C Pathogenic p.((Tyr433Ser)) 83 trio SATB2 chr2:200188564 NM_015265.3 Het De novo (AD) Glass syndrome (OMIM: 612313) Pathogenic PVS1, PS2, PM1, delG c.1504delC PM2 p.(Gln502LysfsTer44) 84 trio SCN1A chr2:166850875 NM_001202435.1 Het De novo (AD) Epilepsy, generalized, with febrile Pathogenic PS2, PM1, PM2, PP2, T>C c.4633A>G seizures plus, type 2 (OMIM: 604403) PP3 p.(Ile1545Val) (AD) Epileptic encephalopathy, early infantile, 6 (Dravet syndrome) (OMIM: 607208) (AD) Febrile seizures, familial, 3A (OMIM: 604403) (AD) Migraine, familial hemiplegic, 3 (OMIM: 609634)

85 trio SCN8A chr12:52159459 NM_014191.3 Het De novo (AD) Cognitive impairment with or Pathogenic PS2, PM1, PM2, PP2, G>A c.2549G>A without cerebellar ataxia PP3, PP5 p.(Arg850Gln) (OMIM: 614306) (AD) Epileptic encephalopathy, early infantile, 13 (OMIM: 614558) (AD) Seizures, benign familial infantile, 5 (OMIM: 617080)

86 duo SERPINC1 chr1:173879999 NM_000488.3 Het Mat (AD,AR) Thrombophilia due to Likely PM2, PM5, T>C c.655A>G antithrombin III deficiency (OMIM: Pathogenic PS4_Moderate, PP2, p.(Asn219Asp) 613118) PP3

87 trio SETD5 chr3:9483407 NM_001292043.1 Het De novo (AD) Mental retardation, autosomal Likely PS2, PM2, PP3, BP1 A>G c.647A>G dominant 23 (OMIM: 615761) Pathogenic p.(Asn216Ser)

88 trio SHANK3 chr22:51160025_5 NM_033517.1 Het De novo (AD) Phelan-McDermid syndrome Pathogenic PVS1, PS2, PM2 1160037delGGGC c.3764_3776del (OMIM: 606232) CCAGCCCCC p.(Arg1255LeufsTer25)

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89 trio SLC2A1 chr1:43394689 NM_006516.2 Het De novo (AD,AR) GLUT1 deficiency syndrome 1, Pathogenic PVS1, PS2, PM2, G>A c.988C>T infantile onset, severe (OMIM: 606777) PP3, PP5 p.(Arg330Ter) (AD) GLUT1 deficiency syndrome 2, childhood onset (OMIM: 612126) (AD) Dystonia 9 (OMIM: 601042) (AD) Stomatin-deficient cryohydrocytosis with neurologic defects (OMIM: 608885)

90 trio SLC2A1 chr1:43394649 NM_006516.2 Het De novo (AD,AR) GLUT1 deficiency syndrome 1, Pathogenic PVS1, PS2, PM2 dupC c.1028dupG infantile onset, severe (OMIM: 606777) p.(Met344HisfsX37) (AD) GLUT1 deficiency syndrome 2, childhood onset (OMIM: 612126) (AD) Dystonia 9 (OMIM: 601042) (AD) Stomatin-deficient cryohydrocytosis with neurologic defects (OMIM: 608885)

91 trio SMAD4 chr18:48604676 NM_005359.5 Het De novo (AD) Myhre syndrome (OMIM: 139210) Pathogenic PS2, PS3, PM1, PM2, A>G c.1498A>G (AD) Juvenile polyposis/hereditary PM5, PP2, PP3, PP5 p.(Ile500Val) hemorrhagic telangiectasia syndrome (OMIM: 175050) (AD) Polyposis, juvenile intestinal (OMIM: 174900) 92 trio SON chr21:34929623 NM_138927.3 Het De novo (AD) ZTTK syndrome (OMIM: 617140) Pathogenic PVS1,PS2, PM2 G>A c.6321+1G>A p.?

93 trio STXBP1 chr9:130440731_1 NM_003165.3 Het De novo (AD) Epileptic encephalopathy, early Pathogenic PVS1, PS2, PM2 30440740dupAAG c.1381_1390dupAAGCC infantile, 4 (OMIM: 612164) CCGGAGC GGAGC p.(Arg464GlnfsTer31)

94 trio STXBP1 chr9:130440777 NM_003165.3 Het De novo (AD) Epileptic encephalopathy, early Pathogenic PVS1, PS2, PM2 C>G c.1427C>G infantile, 4 (OMIM: 612164) p.(Ser476Ter) 95 duo SYNGAP1 chr6:33411735 NM_006772.2 Het Unknown (AD) Mental retardation, autosomal Likely PVS1, PM2 C>T c.3406C>T dominant 5 (OMIM: 612621) Pathogenic p.(Gln1136Ter) 96 trio TBL1XR1 chr3:176767798 NM_024665.4 Het De novo (AD) Mental retardation, autosomal Likely PS2, PM2, PP2, PP3 G>A c.689C>T dominant 41 (OMIM: 616944) Pathogenic p.(Ser230Phe) (AD) Pierpont syndrome (OMIM: 602342)

97 trio TBX1 chr22:19750829 NM_005992.1 Het De novo (AD) DiGeorge syndrome Likely PS2, PM2, PP3 T>C c.476T>C (OMIM: 188400) Pathogenic p.(Leu159Pro) (AD) Tetralogy of Fallot (OMIM: 187500) (AD) Velocardiofacial syndrome (OMIM: 192430)

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98 trio TCF4 chr18:52921925 NM_001243226.2 Het De novo (AD) Corneal dystrophy, Fuchs Pathogenic PVS1, PS2, PS3, G>A c.1459C>T endothelial, 3 (OMIM: 613267) PM2, PS4_Moderate p.(Arg487X) (AD) Pitt-Hopkins syndrome (OMIM: 610954)

99 duo TUBB4A chr19:6495765 NM_001289123.1 Het Unknown (AD) Dystonia 4, torsion, autosomal Pathogenic PS2, PS3, PM1, PM2, C>T c.898G>A dominant (OMIM: 128101) PP2, PP3 p.(Asp300Asn) (AD) Leukodystrophy, hypomyelinating, 6 (OMIM: 612438) 100 trio ZEB2 chr2:145156329 NM_014795.3 Het De novo (AD) Mowat-Wilson syndrome Pathogenic PVS1, PS2, PM2, C>A c.2425G>T (OMIM: 235730) PP3 p.(Glu809Ter)

101 trio ZEB2 chr2:145157206_1 NM_014795.3 Het De novo (AD) Mowat-Wilson syndrome Pathogenic PVS1, PS2, PM2 45157213 c.1541_1548delCGGTA (OMIM: 235730) delCACTACCG GTG p.(Pro514GlnfsTer3) X-LINKED

102 trio DDX3X chrX:41203603 NM_001356.4 Het De novo (XLD,XLR) Mental retardation, X-linked Pathogenic PS2, PM1, PM2, C>T c.976C>T 102 (OMIM: 300958) PM5, PP2, PP3 p.(Arg326Cys) 103 singleton DDX3X chrX:41205795_41 NM_001356.4 Het Unknown (XLD,XLR) Mental retardation, X-linked Pathogenic PVS1, PM1, PM2, 205796delAT c.1535_1536delAT 102 (OMIM: 300958) PM6, PS4_Moderate p.(His512ArgfsTer5)

104 trio IQSEC2 chrX:53263455 NM_001111125.2 Het De novo (XLD) Mental retardation, X-linked 1/78 Pathogenic PVS1, PS2 delG c.4419delC (OMIM: 309530) p.(Ser1474ValfsTer21) 105 trio IQSEC2 chrX:53277294 NM_001111125.2 Het De novo (XLD) Mental retardation, X-linked 1/78 Pathogenic PVS1, PS2, PM2 A>G c.2582+2T>C (OMIM: 309530) p.? 106 trio IQSEC2 chrX:53277371 NM_001111125.2 Het Unknown (XLD) Mental retardation, X-linked 1/78 Likely PS2, PM2, G>A c.2507C>T (OMIM: 309530) Pathogenic PS4_Moderate, PP3 p.(Ala836Val)

107 trio KDM5C chrX:53223464 NM_004187.3 Het De novo (XLR) Mental retardation, X-linked, Pathogenic PVS1, PS2, PM2 C>A c.3895G>T syndromic, Claes-Jensen type p.(Glu1299Ter) (OMIM: 300534)

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108 trio MECP2 chrX:153296860 NM_004992.3 Hemi Mat (XLR) Mental retardation, X-linked, Pathogenic PS3, PM1, PM2, G>A c.419C>T syndromic 13 (OMIM: 300055) PS4_Moderate, PP1, p.(Ala140Val) (XLR) Encephalopathy, neonatal severe PP3, PP5 (OMIM: 300673) (XLR) Mental retardation, X-linked syndromic, Lubs type (OMIM: 300260) (XLD) Rett syndrome (OMIM: 312750) (XL) {Autism susceptibility, X-linked 3} (OMIM: 300496)

109 trio MED12 chrX:70349963 NM_005120.2 Hemi Mat (XLR) Opitz-Kaveggia syndrome VUS PM2, PP3 C>G c.3946C>G (OMIM: 305450) p.(Gln1316Glu) (XLR) Lujan-Fryns syndrome (OMIM: 309520) (XLR) Ohdo syndrome, X-linked (OMIM: 300895)

110 trio NAA10 chrX:153197853 NM_003491.3 Het De novo (XL) ?Microphthalmia, syndromic 1 Pathogenic PS2, PM1, PM2, PP2, A>C c.257T>G (OMIM: 309800) PP3 p.(Leu86Arg) (XLD,XLR) Ogden syndrome (OMIM: 300855) 111 trio PIGA chrX:15339728 NM_002641.3 Hemi De novo (XLR) Multiple congenital anomalies- Likely PS2, PM2, PP3 T>A c.1355A>T hypotonia-seizures syndrome 2 Pathogenic p.(Asp452Va) (OMIM: 300868) (XLR) Paroxysmal nocturnal hemoglobinuria, somatic (OMIM: 300818) 112 duo PIGA chrX:15349685 NM_002641.3 Hemi Mat (XLR) Multiple congenital anomalies- Likely PM1, PM2, PP3, PP5 G>A c.368C>T hypotonia-seizures syndrome 2 Pathogenic p.(Thr123Met) (OMIM: 300868) (XLR) Paroxysmal nocturnal hemoglobinuria, somatic (OMIM: 300818)

113 trio RPS6KA3 chrX:20213249 NM_004586.2 Hemi Mat (XLD) Coffin-Lowry syndrome Likely PM1, PM2, G>A c.340C>T (OMIM: 303600) Pathogenic PS4_Moderate, PP1, p.(Arg114Trp) PP2, PP3 114 trio RPS6KA3 chrX:20179827 NM_004586.2 Hemi De novo (XLD) Coffin-Lowry syndrome Pathogenic PVS1, PS2, PM2, G>A c.1894C>T (OMIM: 303600) PP5 p.(Arg632Ter) 115 trio SMC1A chrX:53410095 NM_001281463.1 Het Unknown (XLD) Cornelia de Lange syndrome 2 Pathogenic PVS1,PM2 dupT c.3053dupA (OMIM: 300590) p.(Arg1019AlafsX26)

Abbreviations:*,proband with two disorders; **,semi-dominant; hemi, hemizygous; het, heterozygous; hom, homozygous; mat, maternal; pat, paternal; AR, autosomal recessive; AD, autosomal dominant; XL, X-linked; XLD, X-linked dominant; XLR, X-linked recessive; VUS, variant of uncertain significance;

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Figure 1. Distribution of variant types for 117 provider-substantiated diagnoses identified by exome sequencing in a pediatric cohort.

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Disease-Associated Mosaic Variation in Clinical Exome Sequencing: A Two Year Pediatric Tertiary Care Experience

Cecelia R Miller, Kristy Lee, Ruthann B Pfau, et al.

Cold Spring Harb Mol Case Stud published online May 5, 2020 Access the most recent version at doi:10.1101/mcs.a005231

Supplementary http://molecularcasestudies.cshlp.org/content/suppl/2020/05/05/mcs.a005231.D Material C1

Published online May 5, 2020 in advance of the full issue.

Accepted Peer-reviewed and accepted for publication but not copyedited or typeset; accepted Manuscript manuscript is likely to differ from the final, published version. Published onlineMay 5, 2020in advance of the full issue.

Creative This article is distributed under the terms of the Commons http://creativecommons.org/licenses/by-nc/4.0/, which permits reuse and License redistribution, except for commercial purposes, provided that the original author and source are credited. Email Alerting Receive free email alerts when new articles cite this article - sign up in the box at the Service top right corner of the article or click here.

Published by Cold Spring Harbor Laboratory Press